Cell viability and genomic stability is jeopardized by DNA base damage, which leads to carcinogenic or lethal events. DNA Base-Excision Repair (BER) enzymes recognize and remove damaged bases and abasic sites that would otherwise cause mutagenic and cytotoxic effects. We aim to understand the structural biochemistry of four ubiquitous enzyme classes, which are both unique and essential to BER and cell viability and furthermore conserved among the three kingdoms of life: 1) Helix-hairpin- Helix DNA glycosylases and glycsylase-apurinic/apyrimidinic (AP) lyases (Endo III class), 2) uracil-DNA glycosylases (UDG/UNG class), 3) major 5'-AP endonucleases (APE-I/Exo III class), and 4) inducible 5'-AP endonucleases (Endo IV/APN-1 class). The proposed structural biochemistry research cycle synergistically couples structural analyses on BER enzymes and enzyme:DNA complexes (Scripps) to detailed biochemical, kinetic, and mutational analyses (SUNY), and is especially critical to deciphering the unifying themes and subtle details of the biochemistry of BER enzymes as several different folds can harbor the constrained loops and pockets acting in BER reactions. We hypothesize that defining the structural biochemistry for BER initiation involves dissecting chemo-mechanical steps that not only provide catalytic specificity, but may also act in the efficient transfer of DNA damage between enzymes. For each BER enzyme class, our experiments are designed to provide a biochemical and structural definition of reaction steps that is testable by designed mutations. Our pyramid approach involves beginning with both thermophilic and mesophilic enzymes plus mutants and then progressively focusing on the most informative constructs. Our strategy of pursuing multiple enzymes and four BER enzyme classes is thus aimed at efficiently achieving a unified understanding with the most appropriate experimental systems. Comparative experiments among BER enzymes and species will test hypotheses from individual structures, reveal unappreciated features, highlight functionally important moieties, better characterize structural motifs and chemical mechanisms, plus aid successful crystallization of enzyme complexes with DNA. Due to the high sequence homology and functional conservation of DNA repair enzymes, this approach should provide structural information on the basis for enzyme activity and specificity, and test hypotheses in the most timely fashion. Importantly, this work focuses upon molecular systems that are directly responsible for the detection and repair of DNA damage underlying cancer susceptibility and initiation.